Timing of the Delta(10,12)-Delta(11,13) double bond migration during ansamitocin biosynthesis in Actinosynnema pretiosum.

The timing of introduction of the unusually placed Delta(11,13) diene system in ansamitocin (AP) biosynthesis was probed by synthesizing optically active potential tri- and tetraketide intermediates as their SNAC thioesters. An AP-nonproducing mutant Actinosynnema pretiosum was complemented by the R enantiomer of the triketide and by the tetraketide with rearranged double bonds, but not by the tetraketide carrying the double bonds in conjugation to the thioester function. The results show that the double bonds are installed in their final positions during processing of the nascent polyketide on module 3 of the asm PKS and that KS4 of the PKS acts as a gatekeeper which accepts only a tetraketide with shifted double bonds as substrate for further processing.

Chemoenzymatic approaches toward dechloroansamitocin P-3.

[reaction: see text] The enantioselective total synthesis of proansamitocin, a key biosynthetic intermediate of the highly potent antitumor agent ansamitocin P-3, is described which bears a diene-ene RCM as the key macrocyclization step. Feeding of proansamitocin to an AHBA block mutant Actinosynnema pretiosum (HGF073) yielded ansamitocin P-3 as well as dechloroansamitocin P-3, the latter also being formed upon fermentation in the presence of 3-amino-5-methoxybenzoic acid.

Determination of the cryptic stereochemistry of the first PKS chain-extension step in ansamitocin biosynthesis by Actinosynnema pretiosum.

The biosynthesis of the antitumor antibiotic, ansamitocin, involves the assembly of a linear octaketide on the ansamitocin (asm) polyketide synthase (PKS), which is then cyclized to proansamitocin and further modified to the final product. In the first chain-extension step on the asm PKS, a stereocenter is generated which is then obliterated in a subsequent double-bond migration. The cryptic configuration at this stereocenter was determined by first synthesizing the two enantiomers of the intermediate diketide as their N-acetylcysteamine (SNAC) thioesters. These were then used to demonstrate that only the R enantiomer complements a 3-amino-5-hydroxybenzoic acid (AHBA) deficient mutant of Actinosynnema pretiosum to restore ansamitocin formation. The low efficiency of complementation by the diketide, compared to AHBA, is due to inefficient loading onto the PKS and not the inhibition of the enzyme. A presumed next chain-extension intermediate-the triketide with an unrearranged double bond-was also synthesized as its SNAC ester, but did not complement the AHBA(-) mutant.

Synthesis of the N-acetylcysteamine thioester of seco-proansamitocin.

[structure: see text] The enantioselective total synthesis of the N-acetylcysteamine thioester of seco-proansamitocin, a key biosynthetic intermediate of the highly potent antitumor agent ansamitocin, is described, which twice utilizes the Nagao acetate aldol reaction, as well as an indium-mediated alkynylation of a benzyl bromide followed by carboalumination. The key step is a Heck reaction between two terminal alkenes for merging the two major fragments.